Insulin dependent diabetes (also termed type-I diabetes and formerly juvenile onset diabetes mellitus) has been classified during the past two decades as a chronic autoimmune disease. In this disorder, cells producing insulin (.beta. cells) within the pancreatic islets are selectively targeted and destroyed by a cellular infiltrate of the pancreas. This inflammatory infiltrate affecting the islets has been termed insulitis. Cells producing insulin comprise the majority of islet cells but less than 2% of the total pancreatic mass (Castano and Eisenbarth, 1990, 1!; Fujita et al., 1982 2!; Foulis et al., 1986 3!). The development of type I diabete can conceptually be divided into six stages, beginning with genetic susceptibility and ending with complete .beta. cell destruction (Eisenbarth, 1986 4!). Stage I is genetic susceptibility, which is a necessary but insufficient condition for development of the disease. A hypothetical triggering event (Stage II) leads to active autoimmunity against .beta. cells (Stage III). In Stage III, the .beta. cell mass is hypothesized to decline and immunologic abnormalities such as autoantibodies directed against insulin and islet cytoplasmic antigens are found. Stimulated insulin secretion is still preserved at this stage. Over a period of years, however, the progressive loss of .beta. cells leads to diminished insulin secretion with intravenous glucose tolerance tests (IVGTT) while the individual is still normoglycemic (Stage IV). Overt diabetes (i.e., diabetes onset or clinical manifestation of disease characterized by hyperglycemia) is Stage V, and can develop years later when approximately 90% of pancreatic .beta. cells are destroyed. In Stage V when overt diabetes is first recognized, some residual insulin production remains (as demonstrated by the presence of the connecting peptide of proinsulin, C peptide, in the serum) but the individual usually requires exogenous insulin for life. Finally, in Stage VI, even the remaining .beta. cells are destroyed and C peptide can no longer be detected in the circulation.
While the initiating factor(s) and specific sequence of events leading to diabetes, including the relative importance of different cell types and cytokines, are still widely debated, a key role is generally recognized for self-antigen reactive T cells (Miller et al., 1988 5!; Harada and Makino, 1986 6!; Koike et al., 1987 7!; Makino et al., 1986 8!). In addition to T lymphocytes, insulitis is characterized by macrophages, dendritic cells (Voorbij et al., 1989 9!) and .beta. cells, which may serve as professional antigen presenting cells (APC). Macrophages may also destroy islet .beta. cells themselves by release of cytokines or free radicals (Nomikos et al., 1986 10!). Thus, autoimmune diabetes relies upon both cellular migration and immune stimulation of newly resident cells.
Cell trafficking to inflammatory sites is regulated by accessory molecules LFA-1, MAC-1 and VLA-4 (Larson and Springer, 1990 11!; Hemler et al., 1990 12!) on the surface of lymphocytes (LFA-1, VLA-4) and macrophages (Mac-1, VLA-4), and by their counterlingands ICAM (for LFA-1 and MAC-1), and VCAM (for VLA-4) which are unregulated by cytokines on vascular endothelium (Larson and Springer, 1990 11!; Lobb, 1992 13!; Osborn, 1990, 14!). In addition, VLA-4 binds to an extracellular matrix component, the CS-1 domain of fibronectin (FN) (Wayner et al., 1989 15!). The relative importance of these pathways, for example, LFA-1 and VLA-4 on lymphocytes or MAC-1 and VLA-4 on monocytes, in controlling cell migration is still a subject of investigation. In vitro data suggest that the differential use of these pathways appears to depend upon the activation status of both the leukocytes and endothelial cells (Shimizu et al., 1991 16!). Their ability to control cell migration to inflammatory sites in vivo has been directly demonstrated with monoclonal antibodies (mAbs) to ICAM, MAC-1 or VLA-4 inhibiting various animal models of disease (Barton et al., 1989 17!, phorbol ester-induced rabbit lung inflammation; Issekutz and Issekutz, 1991 18!, delayed type hypersensitivity; Issekutz, 1991 19!, adjuvant-induced arthritis; Yednock et al., 1992 20!, transfer of experimental allergic encephalomyelitis (EAE); Lobb, 1992 21!, asthma).
ICAM and VCAM are also found on the surface of macrophages and dendritic cells in lymphoid tissues (Dustin et al., 1986 22!; Rice et al., 1990 23!; Rice et al., 1991 24!). Their distribution on these professional APC is consistent with fuinctional data indicating a role for LFA-1 and VLA-4 in T cell activation (Shimuzu et al., 1990 25!, Burkly et al., 1991 26!). However, numerous other receptor-ligand pairs including CD4/MHC class II and CD8/MHC class I (Rudd et al., 1989 27!), CD2/LFA-3 (Moingeon et al., 1989 287!), CD28/B7 (Harding et al., 1992 29!) may also support adhesion or costimulate T cells during T/APC or T/target cell interactions. The specific contributions of these numerous pathways in the development of diabetes is unresolved. Because there are multiple molecular pathways for cell adhesion and T cell activation, it is not possible to predict whether intervention in one or more of these pathways might affect onset or severity of diabetes disease, and, in particular, which of these pathways are crucial or relevant to the disease process.
Antibodies directed to T cells have been utilized in murine and rat models for spontaneous diabetes and adoptive transfer of diabetes to deplete T cells and thus prevent disease (see, e.g., Harada and Makino, 1986 6!, anti-Thy 1.2; Koike et al., 1987 7!, Miller et al., 1988 5! and Shizuru et al., 1988 30!, anti-CD4; Barlow and Like, 1992 31!, anti-CD2; Like et al., 1986 32!, anti-CD5 and anti-CD8). In addition, an antibody directed to the complement receptor type 3 (CR3) molecule or MAC-1 on macrophages has been utilized to prevent macrophage and T cell infiltration of pancreatic tissue in a murine adoptive transfer model of disease (Hutchings et al., 1990 33!). It is unknown whether VLA-4 is relevant to insulitis or to the activity of islet-specific cells after localization in the pancreas.
Current treatment protocols suggested for type I diabetes have included certain immunomodulatory drugs summarized by Federlin and Becker 34! and references cited therein. A long prediabetic period with immunologic abnormalities and progressive .beta. cell destruction suggests it may be possible to halt .beta. cell loss with immune intervention (Castano and Eisenbarth, 1990 1!).
Suggested agents/protocols have included certain immunomodulatory and immunosuppressive agents: levamisol, theophyllin, thymic hormones, ciamexone, antithymocyte globulin, interferon, nicotinamide, gamma globulin infusion, plasmapheresis or white cell transfusion. Agents such as cyclosporin A and azathioprine which impair T cell activation and T cell development, respectively, have been used in clinical trials (Zielasek et al., 1989 35!). The most promising results have been achieved with cyclosporin A (Castano and Eisenbarth, 1990 1!). Federlin and Becker, 1990 34! suggest, however, that cyclosporin A may not be recommended for general or long-term use because of toxic side effects, at least when given in higher doses. Higher doses of cyclosporin, or in combination with other immunosuppressive drugs, or both, have been associated with the development of lymphoma and irreversible kidney damage (Eisenbarth, 1986 4!; Eisenbarth, 1987 36!). Additional studies on other suggested agents are necessary to assess safety and efficacy. Even the cyclosporin A studies show that its efficacy in maintaining remission of diabetes is for one year in about 30-60% of new onset diabetes. Within 3 years, however, remissions are almost invariably lost (Castano and Eisenbarth, 1990 1!). Treatment protocols after onset of disease are particularly problematic, since, for example, at the time diabetes is diagnosed in humans, insulitis has typically progressed already to a loss of more than 80% of the .beta. cells. Thus, it is possible that cyclosporin A may be preventing further .beta. cell destruction, but so few .beta. cells may be present at the onset of the diabetes that they cannot maintain a non-diabetic state over time (Castano and Eisenbarth, 1990 1!). Suppression of insulitis and/or prevention of disease may be more successful if the treatment could start at an earlier phase, i.e., before disease onset.
There are two major prerequisites in order to develop any preventative treatment for diabetes disease: (1) the ability to accurately identify the prediabetic individual and (2) the development of safe, specific and effective preventive treatments. Significant progress has been made in identifying prediabetic individuals, however, much work remains in the development of safe, specific and effective preventive treatments as discussed and reviewed by Eisenbarth and colleagues (see, e.g., Ziegler and Eisenbarth, 1990 37!; Ziegler et al., 1990 38!; Ziegler et al., 1990 39!). It has been possible to identify certain risk factors and at-risk groups for type I diabetes and thus to predict individuals most likely to go on to clinical disease and to estimate the approximate rate of disease onset in these individuals. The ability to identify individuals with susceptibility to diabetes or to predict type I diabetes in the preclinical stage by the combination of genetic (HLA typing), immunological (islet and insulin autoantibodies) and metabolic (first phase insulin secretion to intravenous glucose preceding the development of hyperglycemia) markers makes the identification and use of prophylactic immunotherapeutic drugs and protocols possible during the evolution of the autoimmune disease process when D cell destruction is only partial. To date, there has been little success, however, in treating human diabetes. Generally, because human treatment has been used only after onset of the disease, treatment was followed by a temporary complete or partial remission only in a certain number of patients. Since immunosuppressive mechanisms may prevent insulitis and/or diabetes, there is a need for immunosuppressive components for use in the prediabetic stage. In particular, there is a need for safer and more specifically acting compounds, e.g., monoclonal antibodies, which inhibit entry of effector cells into the pancreas or finction of those cell which may have already entered the islets of Langerhans.
It has now been surprisingly discovered that administering an anti-VLA-4 antibody significantly reduced the incidence of diabetes, in a rodent model of diabetes disease. The NOD mouse model of diabetes is a well established model directly comparable to human type-I diabetes. Using an adoptively transferred disease experimental protocol, irradiated non-diabetic NOD mice were administered splenocytes from spontaneously diabetic NOD mice for the acute transfer of the disease. These splenocytes were treated with anti-VLA-4 antibody before administration and the recipients were also treated for various periods of time after the transfer with anti-VLA-4 antibody.